One focus of the group is to study how organisms sense environmental signals and transduce the signals into changes in gene expression and cell physiology. Specifically, we are examining the E. coli and S. cerevisiae responses to oxidative stress. Reactive oxygen species can lead to the damage of almost all cell components (DNA, lipid membranes, and proteins) and have been implicated as causative agents in several degenerative diseases. Most organisms have an adaptive response to defend against oxidants. For example, treatment of both bacterial and yeast cells with low doses of hydrogen peroxide results in the induction of a distinct group of proteins, the decreased expression of other proteins, and resistance to killing by subsequent higher doses of hydrogen peroxide. In bacterial cells, the key regulator of the inducible defenses against hydrogen peroxide is the OxyR transcription factor. We discovered that OxyR is both the sensor and transducer of the oxidative stress signal; the oxidized but not the reduced form of the purified regulator can activate transcription in vitro. OxyR is activated by the formation of an intramolecular disulfide bond between C199 and C208 and is deactivated by enzymatic reduction by glutaredoxin 1 together with glutathione. Structural studies showed that formation of the C199-C208 disulfide bond leads to a large conformational change. Computational and microarray experiments allowed us to identify many of the genes regulated by OxyR. We now are examining the chemical basis of OxyR sensitivity to hydrogen peroxide and the roles of all of OxyR target genes. Compared to the bacterial response to hydrogen peroxide, less is known about the cellular mechanisms used by higher cells to sense and protect against oxidative damage. To initiate studies of the oxidative stress response in eukaryotes, we constructed isogenic S. cerevisiae strains carrying mutations in known signal transduction pathways and compared the oxidant sensitivities and whole genome expression patterns of these mutants. These studies confirmed that the Yap1 transcription factor is critical for resistance to hydrogen peroxide. We have purified the Yap1 protein and have begun biochemical experiments to characterize this redox-sensitive transcription factor. Mass spectrometry analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629. A stable domain of ~15 kDa was detected upon limited proteolysis of oxidized but not reduced Yap1p. This Yap1p protease resistant domain was purified, and mass spectrometry analysis showed that it was comprised of two separate cysteine-containing peptides of Yap1p. These peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds. Work to determine the structure of this modular redox domain now is underway. A second focus of the group is to identify untranslated, regulatory RNAs and to elucidate their functions. These noncoding RNAs have been shown to have roles in transcriptional regulation, chromosome replication, RNA processing and modification, mRNA stability and translation, and even protein degradation and translocation. We have been characterizing two previously identified E. coli regulatory RNAs, OxyS and 6S RNA. The OxyS RNA, whose expression is induced by OxyR in response to oxidative stress, acts as a global regulator that activates and represses the expression of multiple genes, and as an antimutator that protects cells against DNA damage. Studies of the fhlA and rpoS targets showed that the OxyS RNA represses translation of these genes. We recently found that OxyS RNA action is dependent on the Sm-like Hfq protein and that Hfq functions as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also discovered that the abundant 6S RNA binds and modifies RNA polymerase. Noncoding RNA genes have been missed by most genome annotation; they are usually poor targets in genetic screens and have been difficult to detect by direct sequence inspection. Thus we have been carrying out systematic screens for additional noncoding RNA genes in E. coli. These screens are all applicable to other organisms. One approach based on computer searches of intergenic regions for extended regions of conservation among closely related species has led to the identification of 17 conserved noncoding RNAs. Another screen for noncoding RNAs that coimmunoprecipitate with the Hfq RNA binding protein allowed us to detect six less well conserved RNAs. A third approach of size fraction of total RNA followed by linker ligation and cDNA synthesis has led to the cloning of nine antisense RNAs. Studies to characterize the functions of the newly identified RNAs are ongoing.